Bone marrow monocytes and macrophages from mice lacking βENaC and ASIC2 have a reduced chemotactic migration response and polarization

Abstract The monocyte–macrophage system plays an important role in phagocytosis of pathogens and cellular debris following infection or tissue injury in several pathophysiological conditions. We examined ENaC/ASIC subunit transcript expression and the importance of select subunits in migration of bone marrow derived monocytes (freshly isolated) and macrophages (monocytes differentiated in culture). We also examined the effect of select subunit deletion on macrophage phenotype. BM monocytes were harvested from the femurs of male and female WT and KO mice (6–12 weeks of age). Our results show that α, β, γENaC, and ASIC1‐5 transcripts are expressed in BM macrophages and monocytes to varying degrees. At least αENaC, βENaC, and ASIC2 subunits contribute to chemotactic migration responses in BM monocyte–macrophages. Polarization markers (CD86, soluble TNFα) in BM macrophages from mice lacking ASIC2a plus βENaC were shifted towards the M1 phenotype. Furthermore, select M1 phenotypic markers were recovered with rescue of βENaC or ASIC2. Taken together, these data suggest that βENaC and ASIC2 play an important role in BM macrophage migration and loss of βENaC and/or ASIC2 partially polarizes macrophages to the M1 phenotype. Thus, targeting ENaC/ASIC expression in BM macrophages may regulate their ability to migrate to sites of injury.


| INTRODUCTION
The monocyte-macrophage system is the first responder of the immune system to tissue injury and infection.Monocytes and macrophages are phenotypically mutable.
Monocytes can differentiate into macrophages and macrophages can polarize into a proinflammatory (M1) macrophage phenotype by Interferon Gamma (IFNγ) and Tumor Necrosis Factor Alpha (TNFα), among other cytokines, to promote inflammation and phagocytose damaged tissue.Macrophages can also be polarized into anti-inflammatory (M2) phenotype by select cytokines including Interleukin-4, or -10, leading to inflammation resolution and tissue repair (Murray, 2017).
Circulating monocytes are attracted to and accumulate near the site of injury.Monocytes then invade the tissue and migrate towards the site of injury.Migration is a complex process: the circulating monocyte-macrophages must (1) first sense the chemo-attractants released from the injury site and accumulate, (2) then release enzymes to disrupt the extracellular matrix and cell-to-cell contacts to create a pathway for invasion, and (3) continually disrupt then reform contacts between cell surface molecules and the extracellular environment and reorganize the cytoskeleton as it migrates.As macrophages reach their target, they become polarized, release cytokines and chemokines to attract additional immune cells, then phagocytose damaged tissue and initiate repair processes (Italiani & Boraschi, 2014;Miskolci et al., 2021;Murray, 2017;Orekhov et al., 2019;Santisteban & Iadecola, 2018;Vogel et al., 2014).
To migrate normally, macrophages must be able to interact with their extracellular environment by sensing chemical and mechanical cues.Degenerin ion channels are candidates to integrate these cues in macrophages.Degenerin proteins are known to operate as chemosensors and mechanosensors in a variety of cell types.Acid Sensing Ion Channel (ASIC) subunits form extracellular H + gated cation channels in neurons and smooth muscle (Bianchi & Driscoll, 2002;Drummond, 2021;Kellenberger & Schild, 2002;Syntichaki & Tavernarakis, 2004).Epithelial Na + Channel (ENaC) and select ASIC subunits can act as mechanosensors in neurons, epithelial, endothelial, and smooth muscle cells (Bianchi & Driscoll, 2002;Drummond, 2021;Kellenberger & Schild, 2002;Knoepp et al., 2020;Satlin et al., 2001;Syntichaki & Tavernarakis, 2004).Additionally, several studies suggest certain degenerins are critical to neuritogenesis (Drummond et al., 2006;Fuller et al., 2023;Tao et al., 2022;Zha et al., 2009), a process that is guided by integration of chemical and mechanical cues, similar to migration.Several empirical studies suggest that degenerin family members are expressed in cells of myeloid origin including peripheral blood mononuclear cells, monocytes, splenic and bone marrow derived dendritic cells, and monocyte-macrophage cell line RAW cells (Barbaro et al., 2017;Ertuglu et al., 2024;Nemeth et al., 2022;Ni et al., 2018;Reus-Chavarria et al., 2019).However, which degenerin subunits are expressed in bone marrow derived monocytes and macrophages has not been studied.
A previous study from our laboratory in the RAW monocyte-macrophage cell line suggests that ENaC is required for normal chemotactic migration (Nemeth et al., 2022).Furthermore, polarization to M1 phenotype using IFNγ ± TNFα inhibits of αENaC expression and motility.Surprisingly, loss of ENaC activity induced a subtle morphological and phenotypical change consistent with an M1 macrophage raising the possibility that ENaC inhibition may be a mechanism of polarization.However, whether ENaC or other ASIC subunits play a critical role in bone marrow derived monocyte and macrophage migration and polarization has not been addressed.
In the current investigation, we examined ENaC/ASIC subunit transcript expression and the importance of select subunits in migration of freshly isolated bone marrow derived monocytes and monocytes differentiated into macrophages in culture.We also examined the effect of select subunit deletion and rescue on macrophage polarization status.

| Animals
Male and female wildtype and genetically modified mice (ASIC2 −/− , βENaC m/m , and ASIC2 −/− /βENaC m/m ) were maintained as homozygous mating pairs on C57BL/6 background.Global ASIC2 knockout mice are available through Jackson Labs (RRID: IMSR_JAX:013126).Hypomorphic βENaC (βENaC m/m ) mice are available through the European Mutant Mouse Archive (RRID: IMSR_EM:04574).Global βENaC knockout mice are not available as mice die shortly after birth (McDonald et al., 1999).Our laboratory previously crossed ASIC2 −/− onto βENaC m/m mice to generate ASIC2 −/− /βENaC m/m animals (Kleyman et al., 2009).Animals were housed under 12 h light-dark cycles, provided normal chow (0.3% Na + , Teklad Cat # T.8604) and water ad libitum.All mice were used between 6 and 12 weeks of age.Offspring were genotyped at 3 weeks of age using DNA (DirectTail PCR, Viagen Cat #102-T) isolated from ear punch or tail snip samples, then reconfirmed following tissue isolation using liver samples.All protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.

| RNA extraction
Macrophages on T75 flasks were rinsed with sterile DPBS, scraped into 6 mL ice-cold DPBS (Gibco,, transferred to collection tubes, and centrifuged at 3000 rpm for 20 min, at 4°C.DPBS was removed and cell pellets stored at −80°C.To extract RNA from monocytes, bone marrow samples were pooled from four animals, then stored at −80°C in Trizol (Zymo Research, Cat #R2050-1).Harvested cells were resuspended in 300 μL of cold (~4°C) Trizol, and total RNA was isolated using Quick-RNA Microprep Kit (Zymo Research, Cat #R1050).RNA samples were stored at −80°C.RNA was reverse transcribed using the iScript Advanced cDNA Synthesis Kit (Bio-Rad, Cat # 1725038).Reactions were incubated at 25°C for 5 min, 46°C for 20 min, and 95°C for 1 min.

| Quantitative polymerase chain reaction (qPCR)
qPCR was used to determine (1) mouse α, β, and γENaC, ASIC1-5 gene expression in bone marrow derived macrophages and freshly isolated monocytes and (2) impact of βENaC and ASIC2 loss on expression of macrophage polarization markers (CD68, CD86, CD163, and CD206).Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) gene expression was used as an internal control and to normalize gene expression.TaqMan® primer pair-probe (FAM-MGB) sets were obtained from Applied Biosciences (see Tables 1 and 2).Primer probe sets were tested in 10, 100, and 1000 ng RNA equivalent samples from lung, liver, kidney, and brain samples.Robust PCR products at the expected size were found in the expected tissues.Results for ENaC targets are shown in Nemeth et al. Results for ASIC targets are not shown.Several ASIC2 and ASIC1 primer-probe sets were evaluated.PCR reactions, using BioRad Supermix for Probes (No dUTP, Cat #1863024), were incubated at 95°C for 10 min, followed by 45 cycles of 94°C for 30 s, and 60°C for 1 min, using a ViiA 7 Real-Time PCR System (Applied Biosciences) with MicroAmp Fast Optical 96-Well Reaction Plates [R&D Systems,Inc. (Cat # 4346906)].For all targets in macrophages, we used 100 ng RNA equivalent, except ASIC1 where we used 1000 ng.For samples in freshly isolated monocytes, we used 75 ng RNA equivalent from column purified monocytes.PCR reactions for ENaC, ASIC, and Gapdh were separated on 3%-4% agarose (Fisher Science, Cat # BP1356) gels to ensure that an amplicon of appropriate size was present.GeneRuler Low Range DNA Ladder (Thermo Scientific, Cat # SM0241) was used to estimate size.All samples were run in triplicate.
PCR data were analyzed using QuantStudio™ Real-Time PCR Software v1.6.1.(Applied Biosystems).All amplification curves were examined for logarithmic amplification, baseline stability, noise spikes, and outliers.Trials where the Cycle Threshold (C Th ) values for Gapdh varied greater than 1 cycle among groups were excluded from analysis.C Th values were determined by QuantStudio software using the "relative analysis" setting.To quantify fold expression, we used the delta delta C Th method, where fold expression = 2 −∆∆CT .

| Western blot
We used standard Western blotting techniques to assess αENaC and βENaC expression in freshly isolated bone marrow monocytes.For these experiments, monocytes from two male and two female mice were pooled into a single sample.Cells were lysed in 100 μL of KBO buffer (25 mM Na 3 PO 4 , 300 mM NaCl, 0.5% Triton X-100, and 20 mM octyl glucoside, pH 7.4), then centrifuged at 10,000 rpm for 30 min at 4°C and separated into soluble and insoluble fractions.The soluble fraction was quantified using Biorad Detergent Compatible Bradford Assay Kit (Cat #5000111) and 20 μg was assayed.Protein samples were heated at 100°C for 10 min in 5X Sample Buffer plus DTT (Pierce, Cat #39000) then separated on 7.5% SDS-PAGE (Biorad, Cat # 5671024) and transferred to nitrocellulose (Biorad, Cat #1620112).Blots were blocked using Odyssey Blocking Buffer (LiCor, Cat #927-70001), incubated with rabbit anti αENaC (Stressmark) or rabbit anti-βENaC (Drummond Lab, 1:1000) and incubated overnight, followed by mouse anti-βActin for 1 h.Primary antibody labeling was visualized using Donkey anti-Rabbit IR680RD and Donkey anti-Mouse IR800CW with an Odyssey Infrared Scanner.
To rescue the expression of βENaC or ASIC2 in macrophages from ASIC2 −/− /βENaC m/m animals, we transfected macrophages with full length mouse βENaC or ASIC2a fused to the C-terminus of EGFP or ECFP, respectively (EGFP_βENaC and ECFP_ASIC2a).EGFP alone was used as a control cell line.To enrich the population of cells expressing the truncated or full-length constructs, cells were cultured in the presence of G418 (Roche, Cat # 04727878001) at 1:100 for ~5 passages to generate a stable expressing cell line.EGFP/ECFP expression was confirmed using fluorescence localization.Fluorescence was marginally detected in transfected cells grown in the presence of G418.Un-transfected control cells died within 3 days of exposure.

| Immunolabeling and confocal imaging
For immunolabeling, cells were plated on 8 well chambered cover-glass slides.Near 70% confluency, samples were rinsed with DPBS, fixed in 4% paraformaldehyde for 10 min, then rinsed in DPBS.Samples were treated with the Fc blocking reagent mouse (1:100, Miltenyi, Cat #130-092-575), blocked in 5% donkey serum (Jackson Immuno Research, Cat #017-000-121) for 1 h, then incubated with primary antibodies overnight at 4C.Samples were rinsed then incubated with donkey anti-rabbit Alexa 546 secondary antibodies in 5% donkey serum, rinsed, cover-slipped, and dried.Primary and secondary antibodies, titers, catalog numbers, and RRIDs are provided in Table 3.Samples were imaged using a Leica TCS SP8 confocal microscope, sequential channel scanning at 1024 × 1024 pixels, using a 63X objective under identical conditions for all samples within an experiment.To quantitate fluorescence, regions of interest were drawn around cell borders determined by overlaid signal in 25-30 cells from 2 to 3 images/group by a naive, blinded operator.Fluorescence was normalized to cell area.Images were prepared for presentation in Photoshop and represent original scans.Modifications, where used, were applied to all images within an experiment.Control samples without primary antibody had no signal.

| Expression of ENaC and ASIC transcripts in male and female bone marrow derived macrophages and monocytes
Most ENaC and ASIC transcripts were detected in cultured macrophages derived from bone marrow monocytes and freshly isolated bone marrow monocytes.
Readily detected transcripts include αENaC, βENaC, ASIC2, and ASIC3 in macrophage RNA samples, and αENaC and ASIC2 in freshly isolated bone marrow monocytes (Figure 1).Other ENaC and ASIC transcripts had very high C Th 's (βENaC, ASIC4/5), required more starting material (i.e., 1000 ng of RNA for ASIC1 vs. 100 ng for others), or inconsistent amplification within replicates (γENaC, ASIC1, and ASIC5).C Th ranked lowest to highest: αENaC<ASIC3<βENaC≤ASIC2.With a few exceptions, we found a similar expression profile and C Th ranking for ENaC and ASIC transcripts in freshly isolated monocytes using 75 ng RNA equivalent from monocytes pooled from n = 4 animals of each sex (Figure 1c).ASIC2b was not detected in any samples.Sex differences were identified for αENaC, γENaC, and ASIC4 expression in macrophages and γENaC in monocytes (Figure 1b,c).We confirmed expression of αENaC (~85 kDa) and βENaC (multiple bands ~75 and one ~250 kDa) by Western blotting (Figure 1d).We have observed higher than predicted molecular masses for βENaC in vascular smooth muscle cells (Grifoni et al., 2006;Lu et al., 2022).

| Characterization of bone marrow derived macrophages
We have previously examined the importance of proinflammatory cytokine (IFNγ and TNFα) regulation on macrophage morphology, migration and αENaC expression and wanted to determine if a similar relationship occurs in bone marrow derived macrophages (Nemeth et al., 2022).Similar to RAW cell line, expression of αENaC is abolished and CD86, a marker of activated macrophages (M1), is enhanced following exposure to bone marrow derived macrophages to IFNγ and TNFα.Representative images and quantitative data are provided in Figure 2a.Images in Figure 2b show polarization of bone marrow derived macrophages following treatment with IFNγ, TNFα, or both, characterized by cell loss, and enlargement and flattening

| Role of degenerins in migration of bone marrow derived macrophages
To determine the importance of degenerins in migration of bone marrow derived macrophages, we used 1 μM amiloride, a broad-spectrum inhibitor.As shown in Figure 3, incubating cells with amiloride for 30 min prior to and during migration, reduced chemotactic migration to 56% and  62% of control in female and male macrophages, respectively.Macrophages from female mice showed a greater control migration capacity than macrophages from male mice.To determine the importance of αENaC and γENaC in macrophage migration, we used gene silencing in stably transfected cells from female mice.Silencing of αENaC or βENaC suppressed chemotactic migration by 52% and 51% of control, respectively (Figure 4a).The extent of αENaC and βENaC silencing using quantitative immunolabeling are shown in Figure 4b.Representative immunolabeling images are shown in Figure 4c.Amiloride and gene silencing had a similar impact on migration inhibition.These data support a role for degenerins, including αENaC and βENaC in bone marrow derived macrophage migration.

| Role of select degenerins in freshly isolated bone marrow monocyte migration
To determine if the migratory response in primary bone marrow derived monocytes is also dependent on select degenerins, we studied migration responses of monocytes from βENaC hypomorph (βENaC m/m ) and global ASIC2 knockout (ASIC2 −/− ) mice as these models are available in our laboratory.Mice with global deficiencies in αENaC are not available as they die shortly after birth and γENaC was not detectable in female bone marrow monocytes.Representative images of the underside membrane (Figure 5a,c) and group data (Figure 5b,d) are shown for βENaC m/m and ASIC2 −/− mice, respectively.Migration of bone marrow monocytes was suppressed in βENaC m/m and ASIC2 −/− mice; however, sex and genotype dependent differences were evident.Similar to macrophages, monocytes from female mice had a greater migration response than monocytes from males.In female mice, migration was inhibited to a greater extent in βENaC m/m than ASIC2 −/− mice.In male mice, migration was inhibited to a greater extent in ASIC2 −/− than βENaC m/m mice.Migration responses in monocytes from mice carrying both mutations (ASIC2 −/− x βENaC m/m ) were also attenuated; migration was inhibited to 60% of control in female versus 81% in male monocytes (Figure 6a,b).Comparison of normalized migration responses in freshly isolated bone marrow monocytes from the three genetic models is shown in Figure 6c and suggests loss of both subunits does not have additive effect on monocyte migration inhibition, a finding consistent with the possible formation of heteromeric channels containing at least βENaC and ASIC2.
3.5 | Is there a link between migration and polarization status in bone marrow derived macrophages?
Polarization of macrophages to a proinflammatory phenotype is associated with decreased macrophage mobility and upregulation of select expression markers (Cui et al., 2018;Vogel et al., 2014).Our previously published data in the monocyte-macrophage RAW cell line showed degenerin inhibition with amiloride increased expression of CD86, a proinflammatory marker.However, it was unclear if any degenerins were directly linked to migration and polarization status.To begin to address this issue, we first examined migration and phenotype marker expression in cultured bone marrow derived macrophages from control and βENaC m/m x ASIC2 −/− mice.As expected, we found a loss of βENaC and ASIC2 suppressed chemotactic migration (Figure 7a).Consistent with a pro-inflammatory phenotype, we found an upregulation of the inflammatory M1 marker CD86 message (Figure 7b).CD68 expression, a myeloid origin marker, decreased, and CD206, antiinflammatory M2 marker was not statistically different.
We were unable to detect expression of CD163 by qPCR, F I G U R E 3 Broad spectrum degenerin inhibitor amiloride inhibits migration in male and female bone marrow derived macrophages.Degenerin inhibition with amiloride inhibited migration to 56% and 62% in female and male macrophages, respectively, of control.Baseline migration was greater in female macrophages.Macrophages from male and female animals were treated, migrated, and quantified side-by-side in two independent trials.Data are mean ± SEM and analyzed using two-way ANOVA, followed by Holm-Sidak post hoc test.p values of main factors and their interaction and differences among groups are shown on graph to demonstrate confidence.
an additional M2 marker in control or treated cells (data not shown).Semiquantitative immunolabeling data support the upregulation of CD86 and unchanged CD206 expression as shown by the representative images and group data (Figure 7d,e).Secreted proinflammatory cytokine TNFα in the cell culture media was also increased (Figure 7c).We were unable to detect TGFβ, IL1b, IL6, IL10, IL17, or iNOS in cell culture media.Rescue of βENaC, and ASIC2 to a lesser extent, partially rescued the migration response (Figure 8a).Reintroduction of ASIC2 rescued CD68 expression (Figure 8b).Reintroduction of βENaC rescued CD86 expression as quantified by qPCR and immunolabeling (Figure 8c,d).Reintroduction of βENaC rescued the elevated soluble TNFα in the cell culture media (Figure 8e).Unexpectedly, reintroduction of ASIC2 elevated TNFα above KO-EGFP control.Taken together, these findings suggest βENaC and ASIC2 differentially contribute to macrophage polarization status.

| DISCUSSION
This investigation examined expression of the degenerin proteins, an evolutionarily conserved family of cation ion channels that function in epithelial Na + absorption, extracellular proton sensors, neurotransmitter gated channels, and mechanosensors, in their role in bone marrow derived monocyte-macrophage polarization and migration (Bianchi & Driscoll, 2002;Kellenberger & Schild, 2002;Syntichaki & Tavernarakis, 2004).The major findings of this investigation demonstrate that most degenerins are expressed in bone marrow derived monocytes and macrophages and select degenerins contribute to chemotactic migration and polarization status.Interestingly, we found bone marrow derived monocytes and macrophages from female mice migrate to a greater extent than males.We also found that loss of at least ASIC2 and/or βENaC favors partial polarization towards a proinflammatory phenotype and rescuing expression recovers migratory capacity and phenotypic markers.

| Bone marrow monocytes and derived macrophages have similar degenerin subunit expression pattern
Most degenerin subunits are expressed in bone marrow derived macrophages and freshly isolated monocytes, with minor sex differences.Degenerin message is expressed at relatively low levels with C th 's greater than 30 for all subunits examined.αENaC had the lowest C th , followed by βENaC, ASIC1, ASIC2, and ASIC3.γENaC, ASIC4, and ASIC5 had the highest C th 's.We also found αENaC is the most abundantly expressed degenerin message in monocytes and our previous findings in the RAW cell macrophage cell line F I G U R E 5 Loss of βENaC or ASIC2 inhibits bone marrow monocyte chemotactic migration.Chemotactic migration is attenuated in freshly isolated bone marrow monocytes from (a, b) βENaC hypomorph mice (βENaC m/m , 10-13 weeks of age) or (c, d) ASIC2 global knockout mice (ASIC2 −/− , 7-8 weeks of age).Monocytes were isolated in parallel from an age-matched wildtype and modified animal within a sex then migrated overnight.Mice were used between 7 and 13 weeks of age.These findings suggest (1) migration responses in wildtype mice are greater in female versus males, and (2) βENaC and ASIC2 both contribute to migration, but differentially in the sexes.Loss of βENaC had a larger impact on migration in female versus male, while loss of ASIC2 had a larger impact in males.-- (Nemeth et al., 2022).Our current findings suggest sex differences in macrophage expression of αENaC, γENaC, and ASIC4; C th 's for αENaC and ASIC4 were higher in females.
In bone marrow monocytes, αENaC and ASIC2 were the most easily detected degenerin subunits and we were unable to detect γENaC in females.The physiological relevance of these sex differences is unclear.
4.2 | Is the degenerin expression pattern consistent across monocyte derived cells?
Our findings in bone marrow monocytes and differentiated macrophages share similarities and differences in degenerin expression to peripheral blood mononuclear cells, bone marrow derived dendritic cells, splenic Loss of ASIC2 plus βENaC on bone marrow monocyte chemotactic migration is not additive.Chemotactic migration in freshly isolated bone marrow from mice homozygous for ASIC2 global knockout and βENaC hypomorph alleles (ASIC2 −/− /βENaC m/m , 6-7 weeks).Monocytes were isolated in parallel from an age-matched wildtype and modified animal within a sex then migrated overnight.
Representative images of underside of migration membrane (a) and group data (b) in males are shown.These findings suggest (1) migration responses in wildtype mice are greater in female versus male, and (2) βENaC plus ASIC2 both contribute to migration, but greater impact on female.Loss of βENaC had a larger impact on migration in female versus male, while loss of ASIC2 had a larger impact in males.dendritic cells, and the monocyte-macrophage RAW cell line.Similarities include apparent robust expression of αENaC and weak or lack of expression of γENaC in PBMCs, RAW cells, splenic dendritic cells, and bone marrow monocytes and derived macrophages (Barbaro et al., 2017;Ertuglu et al., 2024;Nemeth et al., 2022).ASIC1-3 are expressed in RAW cells, bone marrow derived dendritic cells, and bone marrow monocytes and derived macrophages (Ni et al., 2018;Tong et al., 2011).In contrast, PBMC monocytes do not express βENaC, ASIC1, or ASIC2 (Ertuglu et al., 2024).Additionally, splenic dendritic cells do not express βENaC (Barbaro et al., 2017).Several factors may account for these variations.First, macrophages have a heterogeneous origin, including bone marrow and tissue resident cells, and undergo different differentiation processes which may contribute to the phenotypic and gene expression differences (Naito, 1993;Zhao et al., 2018).Second, the hormonal and cytokine mileu of the preparation (i.e., blood from hypertensive patients, presence of supplemental F I G U R E 8 Does rescue of ASIC2 or βENaC in bone marrow macrophages from ASIC2 −/− /βENaC m/m mice restore the chemotactic migration response and polarization marker expression?ASIC2 −/− /βENaC m/m (KO) male cell lines were transfected with ECFP_mouse ASIC2 or EGFP_mouse βENaC full length constructs and maintained in the presence of selection antibiotic G418 (except WT control and KO control).(a) Rescue of either ASIC2 or βENaC partially rescues the chemotactic migration response in macrophages lacking ASIC2 and βENaC.Migration data points represent 42-63 FOVs from n = 2 to 3 inserts from three trials.(b) The monocyte origin marker CD68 (b) increased with rescue of ASIC2a and M1 macrophage marker CD86 decreased (c, d), consistent with the decrease in CD68 and increase in CD86 in KO versus WT macrophages (Figure 7).The C Th for GAPDH, CD68, and CD86 in KO-EGFP control samples are within range to detect increases or decreases (20, 20, and 32, respectively).CD163 were not consistently detected in the three replicates from 2 to 5 independent trials.(d).Immunolabeling of CD86-Viobright 515 in KO macrophages rescued with βENaC or ASIC2 are consistent with qPCR findings.Fluorescence signal of CD86 was greater than baseline EGFP/ECFP signal assessed in separate samples (not shown).(e) Soluble TNFα in the media of cultured macrophages (72 h) was decreased in βENaC, but increased in ASIC2 rescued All data are mean ± SEM.Migration data were analyzed using 1-way ANOVA followed by Holm-Sidak post hoc test.Quantitative PCR were analyzed using a 1-way ANOVA followed by Dunnett post hoc test.p values are provided to demonstrate confidence.

| Bone marrow derived macrophages have a similar response to proinflammatory cytokines as the monocyte-macrophage RAW cell line
When exposed to the proinflammatory cytokines IFNγ and TNFα, bone marrow derived differentiated macrophages exhibited morphological changes (enlargement, flattening, and reduced circularity), cell loss, reduced αENaC expression, and suppressed chemotactic migration similar to our previous findings in the RAW cell line (Nemeth et al., 2022).In contrast to the RAW cell line, bone marrow derived macrophages required a survival factor such as CSF1 or CSF2 to proliferate in culture.

| Degenerins are required for normal bone marrow derived macrophage and monocyte chemotactic migration
We used several approaches to determine the importance of degenerins in migration.We initially used the broad spectrum degenerin inhibitor amiloride and found 1 μM inhibited migration to 56% and 62% in female and male macrophages, respectively, of control.The IC 50 of amiloride for ENaC is ~0.1 μM and 10 μM for ASIC channels, the concentration of amiloride used in the current study, favors inhibition of ENaC channels and ASIC channels are blocked to a lesser extent (Lingueglia & Lazdunski, 2013).Chemotactic migration in macrophages was inhibited using gene silencing approaches.We also examined the importance of select degenerins in macrophage migration using dominant negative directed gene silencing (αENaC and βENaC) and genetically modified (ASIC2 −/− × βENaC m/m ) animals.We found chemotactic migration of macrophages were inhibited to a similar extent (~50%), regardless of the approach.Since degenerins are known to form heteromeric channels, namely, it likely that some degenerins may associate to form multimeric channels in macrophages as well.This possibility is supported by our findings in freshly monocytes; while the loss of βENaC or ASIC2 alone had differing impacts on migration inhibition, they were not additive.
4.5 | What is the role of other degenerins ASIC1 and ASIC3 in migration?
Although we detected expression of other ASIC channels in addition to ASIC2, we did not directly examine their role in migration.Other reports have addressed the role of ASIC1 using psalmotoxin, an inhibitor of ASIC1 homomeric, but not heteromeric, channels (Ni et al., 2018) on wound healing migration.The tendency for ASIC subunits to form heteromeric channels with no selective pharmacologic blocking or activating agents makes this challenging without gene silencing or knockout models.Extracellular proton gated ASIC-like currents are present in dendritic cells; however, their molecular identity is undetermined (Tong et al., 2011).While ASIC1 and ASIC2 may be positive regulators of migration, other ASIC channels may not play a similar role.Not all ASIC subunits contribute similarly to vascular smooth muscle cell and glial migration (Grifoni et al., 2008;Kapoor et al., 2009;Vila-Carriles et al., 2007).

| Greater migration response in monocytes and macrophages from females compared to males
We consistently found that the chemotactic migration response in control animals were greater macrophages and monocytes from female versus male mice, a novel finding not previously reported to our knowledge.The mechanisms underlying this difference are unclear but may influenced by female sex steroids on ENaC expression as sexual dimorphism in ENaC expression in renal tubular tissue and endothelial cells (Gambling et al., 2004;Padilla et al., 2019;Veiras et al., 2017).
4.7 | Why isn't there a more profound polarization following loss of ASIC2 and βENaC?
In our current and previous study using pharmacological ENaC inhibition, we found a modest polarization to the M1 phenotype (Nemeth et al., 2022).The current study provides direct evidence that βENaC and ASIC2 contribute to M1 polarization as rescue of either subunit differently contribute to normalization of CD86 expression and TNFα secretion in the macrophages lacking ASIC2 and βENaC.This finding, combined with our finding that polarization with IFNγ/TNFα suppresses at least αENaC expression (in RAW cells and bone marrow derived macrophages), suggests silencing of βENaC and ASIC2 is part of the proinflammatory cytokine-initiated polarization signaling (Nemeth et al.,Figure 2a,b).The impact of proinflammatory cytokines of expression on expression of other subunits have not been examined.The proinflammatory cytokines, including IFNγ, TNFα, lipopolysaccharide, generally activate transcription factors (STAT1/3/6) which activate other signaling factors such as NOS or NFkβ (Murray, 2017;Orekhov et al., 2019).This raises the possibility that proinflammatory cytokine mediated inhibition of degenerins may be a mechanistic component of polarization patterning.
Mammalian degenerin channels are activated by multiple factors including neurohumoral (β2 adrenergic and mineral-glucocorticoids), extracellular protons (ASIC channels), intracellular proteases (furin), and mechanical forces (shear stress and strain) (Fronius, 2013).αβγENaC channels are constitutively active channels that are sensitive to changes in extracellular Na + (heterologous expression systems and renal collecting duct cells) and can be gated by shear stress (endothelial cells and renal collecting duct cells) and strain (vascular smooth muscle cells) (Awayda et al., 1995;Barbaro et al., 2017;Carattino et al., 2004;Jernigan & Drummond, 2006;Knoepp et al., 2020;Mano & Driscoll, 1999;Shi et al., 2013).Several ASIC channels are required for mechano-dependent processes in touch receptors, baroreceptors, and VSMCs (Gannon et al., 2008(Gannon et al., , 2015;;Lu et al., 2009Lu et al., , 2022;;Price et al., 2000).Additionally, degenerin proteins have large extracellular domains (~300-400 amino acids) that form an antennae like structure that is likely to interact with the extracellular matrix (Welsh et al., 2002).Migrating cells are exposed to mechanical forces as monocytes "roll" along the intravascular space and as extracellular matrix contacts are disrupted then reformed to accommodate cell movement through tissue towards site of injury.Additionally, changes in cell volume and shape (forming leading and trailing edges) occur during migration.Thus, it is possible degenerins may participate in the signaling of mechanical forces may.We hypothesize that ENaC-ASIC may play a role in sensing mechanical forces through interactions with the extracellular matrix and/or cell volume to contribute to signaling underlying migration.

| SIGNIFICANCE AND PERSPECTIVES
Our findings suggest that transcripts for degenerin channels are expressed in bone marrow monocytes and macrophages and certain degenerins are required for monocyte/macrophage migration, a fundamental phenotype, which is dependent upon polarization status.Degenerins may act as sensors of the chemical and mechanical features of the extracellular environment to regulate M1 polarization and migration.How loss of degenerin function may affect phagocytosis of damaged tissue and polarization to the reparative M2 phenotype is unclear and will be determined by future studies.

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I G U R E 1 qPCR detection of α, β, γENaC, and ASIC1-5 transcript expression in cultured bone marrow derived macrophages and freshly isolated monocytes from male and female mice.Macrophages were cultured in the presence of 20 ng/mL of CSF1.(a) PCR reactions from macrophages were separated on 3%-4% agarose gels to determine if amplicon of expected size was present (identified by arrowhead in samples with >1 product).100 ng RNA template equivalent was used for all reactions except ASIC1, where 1000 ng was used.Three primer pair-probe sets were tested on ASIC2 and ASIC1.The primer pair-probe sets shown amplified a band at the expected size, in addition to 1-2 other bands.(b) Macrophage C th 's individual ENaC and ASIC transcripts from bone marrow derived macrophages 3 replicates in n = 2 trials.Thresholds at or near 35 cycles were consistently identified in all replicates for αENaC and ASIC3.Thresholds less than 39 cycles were identified for βENaC and ASIC2.γENaC amplified in only 1/6 and 4/6 replicates in male and female samples, respectively.(c) Bone marrow derived freshly isolated monocytes C th 's for individual ENaC and ASIC subunits using 75 ng RNA template equivalent.Samples from four animals were pooled.(d) Bone marrow derived freshly isolated monocyte Western blot detection of αENaC and βENaC.Samples were pooled from four animals.*Indicates statistical difference between male and female at p < 0.05 using an Uncorrected Fisher LSD Test. .Exposure to IFNγ or IFNγ plus TNFα, but not TNFα alone, inhibited migration of bone marrow derived macrophages.Representative images of the underside of the migration membrane and quantitative data are shown (Figure2c,d).These findings suggest that bone marrow derived macrophages generally behave similarly to IFNγ and TNFα in regards to αENaC expression, morphological changes, and migration responses.

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Proinflammatory cytokines IFNγ + TNFα on CD86/αENaC expression and chemotactic migration in bone marrow macrophages.Cells (female) were grown in the presence of CSF1 for 7 days.IFNγ (10 ng/mL) and TNFα (10 ng/mL) were added for the last 48 h and 24 h, respectively.(a) Representative images (left) and group data (right) showing αENaC expression is inhibited while CD86 expression is upregulated with polarization to M1 macrophages following IFNγ + TNFα treatment (n = 73-75 cells from n = 3 images per group).Data analyzed using a two-tailed, Mann-Whitney test.(b) Light microscopy images of bone marrow macrophages treated with TNFα ± IFNγ prior to migration.Cells exhibit morphological changes consistent with polarization: Cells become flatter, less round and enlarged with processes.(c) Representative images of the underside of migration membranes where cells are stained blue.(d) Group data showing IFNγ, with or without TNFα, suppressed 24 h migration.TNFα had a negligible impact on migration.Data are mean ± SEM and analyzed using independent, two-tailed t-tests (a, b) or one-way ANOVA, followed by Holm-Sidak post hoc test (d).p values of differences among groups are shown on graph and demonstrate confidence.

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Silencing of αENaC and βENaC in bone marrow macrophages inhibits chemotactic migration.(a) Plots showing chemotactic migration responses in wildtype (WT) female cells stably transfected with EGFP_ αENaCW112X or EGFP_βENaCI41X are suppressed greater than 50% of control (EGFP).Cells were maintained in the presence of G418 (1:100).Group data (b) and representative images (c) of semiquantitative immunolabeling shows αENaC and βENaC expression (Donkey anti-Rabbit Alexa 546) is suppressed in transfected cells.Migration data represent seven FOVs, from three inserts, in two independent trials.Immunolabeling represent n = 50-100 cells from n = 2-4 images.Antibodies are directed to sequences downstream of the dominant-negative construct.All data are mean ± SEM and analyzed using Brown Forsyth-Welch Test followed by Dunnett post hoc test.p values are provided on graph and demonstrate confidence.
Normalized migration data are shown in Figure Representative images are shown in panels a and c and group data are shown in panels b and d.Data are mean ± SEM and represent seven FOVs from three insets (n = 21) and were analyzed using two-way ANOVA followed by Holm-Sidak post hoc test.p values of main factors and their interaction and differences among groups are shown on the graph and demonstrate confidence.
(c).Normalized migration data in monocytes from βENaC m/m , ASIC2 −/− , and ASIC2 −/− /βENaC m/m mice are shown.Both data sets are presented as mean ± SEM and represent seven FOVs from three insets (n = 21, except wildtype control in C where n = 21 FOVs from n = 9 inserts) and were analyzed using 2-way ANOVA followed by Holm-Sidak post hoc test.p values of main factors and their interaction and differences among groups are shown on the graph and demonstrate confidence.

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I G U R E 7 Bone marrow macrophages from mice lacking ASIC2 plus βENaC (ASIC2 −/− /βENaC m/m , KO) are polarized towards an M1 phenotype.(a) Migration of bone marrow derived macrophages from ASIC2 −/− /βENaC m/m mice are inhibited to 55% and 65% of WT control cells from females and males, respectively.Data were analyzed using 2-way ANOVA followed by Holm-Sidak post hoc test.(b) Fold expression of monocyte/macrophage marker message in cultured bone marrow macrophages from males.The myeloid origin marker CD68 was decreased and M1 macrophage marker CD86 was upregulated in KO cells.The M2 marker CD206 was not significantly elevated.CD163, another marker of M2 macrophages, did not amplify in any samples.(c) Media soluble TNFα, released from M1 macrophages, was elevated in KO cell culture media.Samples were obtained from two wells from three different cell lines.Data in Panels b and d were analyzed using independent/unpaired, 2-tailed t-tests.Representative images (d) and group data (e) from semiquantitative immunolabeling of CD86 and CD206 in cells show CD86, but not CD206, are elevated in KO cells.each data point represents a cluster of cells, n = 5-6 cell clusters from n = 3 images.Fluorescence is normalized to cell area.Data in panels e and f analyzed using 1-way ANOVA followed by the Holm-Sidak post hoc test.These findings suggest bone marrow macrophages from mice lacking βENaC and ASIC2 are polarized towards the M1 phenotype.All data are mean ± SEM.P values are provided to demonstrate confidence.
factors, and serum content in cell culture environment) likely influences expression of the degenerin expression pattern.The robust expression of serum glucocorticoid kinase 1, an important regulator of ENaC expression, in PBM monocytes and dendritic cells and the sensitivity of monocyte-macrophage degenerin expression to proinflammatory cytokines are two findings that are consistent with this possibility(Ertuglu et al., 2024;Nemeth et al., 2022).
TaqMan Gene Expression Assay Primer-Probe Pairs for mouse ENaC Subunits.
T A B L E 1Note: ASIC1 Mm01306004 did not detect a band at predicted size.ASIC1 Mm01306001 did not detect band at predicted size.ASIC2 Mm00475691 detected 2 bands, 1 at predicted size.ASIC2 Mm01304217 did not detect band at predicted size.ASIC2b was detected in 1 out of 4 different macrophage samples, at predicted size.a Multiple Primer-Probe pairs were tested for ACCN2, ACCN1.b The blue highlighted "Assay Name" was used in the current study.a C T not detected in any samples up to 45 cycles.T A B L E 2 TaqMan Gene Expression Assay Primer-Probe Pairs for Macrophage (M1/M2) Polarization.

10 | Macrophage activation/ proinflammatory cytokine treatment
night.Insert upper surfaces were scraped with a cotton swab to remove unmigrated cells, gently rinsed in DPBS, fixed in 70% ice cold methanol for 10 min, stained with hematoxylin 10 min, then thoroughly rinsed under gently running water for 10 min.Cells on the bottom surface of the insert were counted on a Nikon Eclipse 200 inverted microscope using a 20× objective.Migration was quantified from seven fields of view/insert, from n = 3 inserts, for each group.Light microscopy images of representative fields of view were collected using a Nikon Eclipse 200 inverted microscope with 20× objective, CoolSnap Color camera, and Metamorph software.2.2.11 | Statistical analysisData sets with similar variability were analyzed using 2-tailed, independent t-tests, one, two or three-way ANOVA, using Prism 9 software, and presented as mean ± SEM.Data that did not pass a normal distribution test were analyzed using a Mann-Whitney Test (two groups) or Brown Forsyth/Welch Test (>2 groups).Post hoc comparisons to a control group use a Dunnett test, while comparisons among multiple groups used the Holm-Sidak test.Bar graphs with individual data points are provided.Figure legends identify sample sizes and specific analyses applied.A value of p ≤ 0.05 is statistically significant.Result "trends" are also reported to avoid erroneous true/false conclusions based on bright-line rules.Select p values are provided to demonstrate confidence.